Detection of Crimean-Congo hemorrhagic fever, Hanta, and sandfly fever viruses by real-time RT-PCR.

The development of sensitive and specific nucleic acid diagnostic assays for viral pathogens is essential for proper medical intervention. This chapter describes four fluorescence-based PCR assays to detect the Crimean-Congo Hemorrhagic Fever (CCHFV), Andes (ANDV), Hantaan (HANV), and Sandfly Fever Sicilian (SFSV) Viruses. These assays are based on species-specific hydrolysis probes targeting the nucleocapsid protein gene for CCHFV and SFSV and the glycoprotein gene for ANDV and HANV. All four assays were optimized for LightCycler 2.0 (Roche Diagnostics, Indianapolis, IN) or Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.; Idaho Technology Inc., Salt Lake City, UT). The assays were evaluated using the protocols described in the Subheading 3. The limits of detection were approximately 5, 2, 2, and 5 plaque-forming units (PFUs) for CCHFV, ANDV, HTNV, and SFSV assays, respectively. The sensitivity and specificity of the assays were evaluated with test panels that consisted of 20-60 known positive and 30-135 known negative samples, representing 7-34 genetically diverse bacterial and viral species. The CCHFV assay detected 59 out of the 60 positive samples and no false positives, resulting in 98.3% sensitivity at LOD of 5 PFU and 100% specificity. The ANDV and HTNV assays correctly identified all the positive samples with no false positive reactions; therefore, the sensitivity and specificity of these assays were determined to be 100% at LOD of 2 PFU. The SFSV assay missed three positive samples and cross-reacted with one of 48 negative samples, resulting in 95% sensitivity at LOD of 5 PFU and 98% specificity.

Inhibition of monkeypox virus replication by RNA interference.

The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV), Vaccinia (VACV), monkeypox (MPV) and Variola (VARV) viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi) as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA). Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R) or an important gene in viral entry (E8L), inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.